Mammalian pulmonary surfactant is a mixture of proteins (10%) and lipids (90%) including the major lipid component dipalmitoylphosphatidylcholine (Zuo Y Y, et al., Biochim Biophys Acta (2008) 1778:1947-77). The main function of the pulmonary surfactant is to ensure minimal surface tension within the lung to avoid collapse during respiration. Furthermore, by interacting with inhaled pathogens, the pulmonary surfactant also participates in host defense (Clements J A. Am Rev Respir Dis (1977) 115:67-71). Pulmonary surfactant deficiency is, therefore, associated with pulmonary diseases such as asthma, bronchiolitis, respiratory distress syndrome (RDS), cystic fibrosis, and pneumonia (Griese M. Eur Respir J (1999) 13:1455-76). Surfactant formulations are indicated for the treatment of RDS, which affects ˜1.5 million premature babies globally every year. Respiratory distress syndrome is a major pulmonary surfactant deficiency disease caused by the structural immaturity of the lungs in premature infants, which makes it difficult to breathe, inhibits gas exchange, and promotes alveolar collapse (Notter R H. Lung Surfactants. Basic Science and Clinical Applications. New York, N.Y.: Marcel Dekker Inc.). However, treatment becomes more difficult if the lungs are infected or if there are inflammatory or oxidative complications, because current surfactant preparations lack surfactant protein D (SP-D). The successful treatment of complex pulmonary diseases, therefore, requires the production of surfactant formulations whose composition matches natural pulmonary surfactant as closely as possible (Robertson B, et al., Biochim Biophys Acta (1998) 1408:346-61).
SP-D has a role in the pulmonary innate immune system by providing anti-inflammatory and antimicrobial activities that address chronic pulmonary diseases such as asthma, cystic fibrosis, and smoking-induced emphysema (Clark H, et al., Immunobiology (2002) 205:619-31). Data based on premature newborn lambs suggest that the administration of ˜2-3 mg/kg of recombinant human SP-D in combination with 100 mg/kg Survanta® (a natural surfactant available in USA) is more effective than Survanta® alone for the prevention of endotoxin shock and the reduction of lung inflammation caused by ventilation (Ikegami M, et al., Am J Respir Crit Care Med (2006) 173:1342-7; Sato A, et al., Am J Respir Crit Care Med (2010) 181:1098-105).
Traditionally, SP-D has been isolated from the supernatant of bronchoalveolar lavage or amniotic fluid, but most SP-D is lost during purification, in part due to the hydrophilic properties of SP-D (Dodagatta-Marri E, et al., Methods Mol Biol (2014) 100:273-90). The use of natural SP-D to supplement pulmonary surfactant formulations can ensure therapeutic efficiency because higher-order multimerization in the endogenous surfactant increases the number of SP-D-binding sites to carbohydrate ligands on the surface of pathogens, achieving potent bacterial and viral agglutination effects (White M, et al., J Immunol (2008) 181:7936-43). The appropriate oligomerization state is also required for receptor recognition and receptor-mediated signal transduction for modulation of the host immune response (Yamoze M et al., J Biol Chem (2008) 283:35878-35888) as well as for maintenance of surfactant homeostasis (Zhang L et al., J Biol Chem (2001) 276:19214-19219).
The low SP-D yields and variable oligomerization states make it difficult to use natural sources for the production of pharmaceutical SP-D (Strong P, et al., J Immunol Methods (1998) 220:139-49). To overcome some of these limitations, recombinant SP-D can be produced in microbes or mammalian cell lines, potentially offering a large-scale platform for the production of homogeneous recombinant SP-D formulations. However, it is challenging to express recombinant human SP-D (rhSP-D) to levels sufficient for a commercial campaign in commonly used mammalian cell lines because the protein is not synthesized efficiently and yields are typically <2 mg of purified protein per liter. Although yields tend to be higher in non-mammalian systems, expression of only a truncated variant of SP-D has been attempted in systems such as yeast or bacteria which have the disadvantage of either not producing the glycosylated form of the protein, or not producing the protein with a human glycosylation pattern (Salgado D, et al., Front Immunol (2014) 5:623, doi: 10.3389/fimmu.2014.00623). Furthermore, it has not been possible to date to control the variability in oligomerization states seen with recombinant and natural human SP-D. Unless the expression system can reproducibly produce rhSP-D with consistently stably levels of the higher-order multimerization states observed in natural SP-D, there is a potential for reduced efficacy of such preparations.